High intensity focused ultrasound (HIFU) has emerged as a new and promising treatment modality for a broad variety of cancers. MRI-guided HIFU (MRgHIFU) allows for the non-surgical, precise ablation of tissue masses in almost all internal organs. MRgHIFU is advantageous in treating patients with unresectable cancers or with poor physical condition for surgery. Moreover, unlike radiation and chemotherapy, HIFU can be applied repetitively without accumulating systemic toxicity. HIFU has 3 main approaches for mass ablation: (i) continuous-wave ablation, rapidly resulting in high temperatures (>60C), necrotic coagulation of the tissue, and protein denaturation; (ii) pulsed-wave ablation, leading to hyperthermia (?50C) and heat-stress induced apoptosis, and (iii) histotripsy, mechanical disruption without changes in temperature. Current anti-cancer approaches use high-heat ablation as they aim for the shortest treatment time for complete tumor ablation. While this approach eliminates the targeted tumor, it does not necessarily result in the development of adaptive antitumor responses that are required for the elimination of metastases and prevention of recurrences. Induction of adaptive anti-tumor responses requires the uptake of dying/dead tumor cells by DCs followed by tumor antigen presentation to both CD8+ and CD4+ T cells in a stimulatory context. Uptake of dying/dead cells is generally a tolerogenic process in order to prevent development of autoimmune response upon normal tissue turnover. However, specific types of cell death result in the release of distinct Death Associated Molecular Patterns (DAMPs) that act on DCs and instill an immunostimulatory phenotype and promote T cell priming to cell-associated antigens. It is likely that the 3 HIFU approaches have distinct effects on the stability, configuration, and release of both the DAMPs and tumor antigens. However, there is currently is no information how the 3 different HIFU approaches affect the development of the adaptive anti-tumor response. The overall objective of this application is to generate quantitative and qualitative insight into the development of adaptive responses to heat-stable and heat-labile tumor antigens after different MRgHIFU treatments that can be used to optimize HIFU strategies in different clinical settings. We will use an in vivo tumor ablation model to determine the effect of different MRgHIFU approaches on the magnitude, phenotype, polyfunctionality, memory development, and protective capacity of tumor-specific CD8+ and CD4+ T cells. As many, if not most, HIFU protocols are still under development, the outcomes of our pre-clinical studies have high translational potential as they will provide the first immunological evidence-based rationale for the design of MRgHFU treatments. Moreover, they will provide insight into the kinetics of the adaptive response that can be used to determine the most efficacious window for combinational treatments such as immune- checkpoint interference or additional chemotherapy.